The present invention relates to novel antagonists of endothelin useful as pharmaceutical agents, to methods for their production, to pharmaceutical compositions which include these compounds and a pharmaceutically acceptable carrier, and to pharmaceutical methods of treatment. More particularly, the novel compounds of the present invention are antagonists of endothelin useful in treating elevated levels of endothelin, acute and chronic renal failure, hypertension, myocardial infarction, metabolic, endocrinological, neurological disorders, congestive heart failure, endotoxic shock, subarachnoid hemorrhage, arrhythmias, asthma, preeclampsia, Raynaud's disease, percutaneous transluminal coronary angioplasty and restenosis, angina, cancer, pulmonary hypertension, ischemic disease, gastric mucosal damage, ischemic bowel disease, and diabetes.
Endothelin-1 (ET-1), a potent vasoconstrictor, is a 21 amino acid bicyclic peptide that was first isolated from cultured porcine aortic endothelial cells. Endothelin-1, is one of a family of structurally similar bicyclic peptides which include; ET-2, ET-3, vasoactive intestinal contractor (VIC), and the sarafotoxins (SRTX's). The unique bicyclic structure and corresponding arrangement of the disulfide bridges of ET-1, which are the same for the endothelins, VIC, and the sarafotoxins, has led to significant speculation as to the importance of the resulting induced secondary structure to receptor binding and functional activity. ET-1 analogues with incorrect disulfide pairings exhibit at least 100-fold less vasoconstrictor activity. The flexible C-terminal hexapeptide of ET-1 has been shown to be important for binding to the ET receptor and functional activity in selected tissues. Additionally, the C-terminal amino acid (Trp-21) has a critical role in binding and vasoconstrictor activity, since ET[1-20] exhibits approximately 1000-fold less functional activity.
Endothelin is involved in many human disease states.
Several in vivo studies with ET antibodies have been reported in disease models. Left coronary artery ligation and reperfusion to induce myocardial infarction in the rat heart, caused a four- to sevenfold increase in endogenous endothelin levels. Administration of ET antibody was reported to reduce the size of the infarction in a dose-dependent manner (Watanabe, T., et al, "Endothelin in Myocardial Infarction," i Nature (Lond.) 344:114 (1990)). Thus, ET may be involved in the pathogenesis of congestive heart failure and myocardial ischemia (Margulies, K. B., et al, "Increased Endothelin in Experimental Heart Failure," Circulation 82:2226 (1990)).
Studies by Kon and colleagues using anti-ET antibodies in an ischemic kidney model, to deactivate endogenous ET, indicated the peptide's involvement in acute renal ischemic injury (Kon, V., et al, "Glomerular Actions of Endothelin In Vivo," J. Clin. Invest. 83:1762 (1989)). In isolated kidneys, preexposed to specific antiendothelin antibody and then challenged with cyclosporine, the renal perfusate flow and glomerular filtration rate increased, while renal resistance decreased as compared with isolated kidneys preexposed to a nonimmunized rabbit serum. The effectiveness and specificity of the anti-ET antibody were confirmed by its capacity to prevent renal deterioration caused by a single bolus dose (150 pmol) of synthetic ET, but not by infusion of angiotensin II, norepinephrine, or the thromboxane A.sub.2 mimetic U-46619 in isolated kidneys (Perico, N., et al, "Endothelin Mediates the Renal Vasoconstriction Induced by Cyclosporine in the Rat," J. Am. Soc. Nephrol. 1:76 (1990)).
Others have reported inhibition of ET-1 or ET-2-induced vasoconstriction in rat isolated thoracic aorta using a monoclonal antibody to ET-1 (Koshi, T., et al, "Inhibition of Endothelin (ET)-1 and ET-2-Induced Vasoconstriction by Anti-ET-1 Monoclonal Antibody," Chem. Pharm. Bull., 39:1295 (1991)).
Combined administration of ET-1 and ET-1 antibody to rabbits showed significant inhibition of the BP and renal blood flow responses (Miyamori, I., et al, "Systemic and Regional Effects of Endothelin in Rabbits: Effects of Endothelin Antibody," Clin. Exp. Pharmacol, Physiol., 17:691 (1990)).
Other investigators have reported that infusion of ET-specific antibodies into spontaneously hypertensive rats (SHR) decreased mean arterial pressure (MAP), and increased glomerular filtration rate and renal blood flow. In the control study with normotensive Wistar-Kyoto rats (WKY) there were no significant changes in these parameters (Ohno, A. "Effects of Endothelin-Specific Antibodies and Endothelin in Spontaneously Hypertensive Rats," J. Tokyo Women's Med. Coll., 61:951 (1991)).
In addition, elevated levels of endothelin have been reported in several disease states (see Table I below).
Burnett and co-workers recently demonstrated that exogenous infusion of ET (2.5 ng/kg/mL) to anesthetized dogs, producing a doubling of the circulating concentration, did have biological actions (Lerman, A., et al, "Endothelin has Biological Actions at Pathophysiological Concentrations," Circulation 83:1808 (1991)). Thus heart rate and cardiac output decreased in association with increased renal and systemic vascular resistances and antinatriuresis. These studies support a role for endothelin in the regulation of cardiovascular, renal, and endocrine function.
In the anesthetized dog with congestive heart failure, a significant two- to threefold elevation of circulating ET levels has been reported (Cavero, P. G., et al, "Endothelin in Experimental Congestive Heart Failure in the Anesthetized Dog," Am. J. Physiol. 259:F312 (1990)), and studies in humans have shown similar increases (Rodeheffer, R. J., et al, "Circulating Plasma Endothelin Correlates With the Severity of Congestive Heart Failure in Humans," Am. J. Hypertension 4:9A (1991)). When ET was chronically infused into male rats, to determine whether a long-term increase in circulating ET levels would cause a sustained elevation in mean arterial blood pressure, significant, sustained, and dose-dependent increases in mean arterial BP were observed. Similar results were observed with ET-3 although larger doses were required (Mortenson, L. H., et al, "Chronic Hypertension Produced by Infusion of Endothelin in Rats," Hypertension, 15:729 (1990)).
The distribution of the two cloned receptor subtypes, termed ET.sub.A and ET.sub.B, have been studied extensively (Arai, H., et al, Nature 348:730 (1990), Sakurai, T., et al, Nature 348:732 (1990)). The ET.sub.A, or vascular smooth muscle receptor, is widely distributed in cardiovascular tissues and in certain regions of the brain (Lin, H. Y., et al, Proc. Natl. Acad. Sci. 88: 3185 (1991)) . The ET.sub.B receptor, originally cloned from rat lung, has been found in rat cerebellum and in endothelial cells, although it is not known if the ET.sub.B receptors are the same from these sources. The human ET receptor subtypes have been cloned and expressed (Sakamoto, A., et al, Biochem. Biophys. Res. Chem. 178:656 (1991), Hosoda, K., et al, FEBS Lett. 287:23 (1991)). The ET.sub.A receptor clearly mediates vasoconstriction and there have been a few reports implicating the ET.sub.B receptor in the initial vasodilatory response to ET (Takayanagi, R., et al, FEBS Lett. 282:103 (1991)) . However, recent data has shown that the ET.sub.B receptor can also mediate vasoconstriction in some tissue beds (Panek, R. L., et al, Biochem. Biophys. Res. Commun. 183(2):566 (1992)).
Comparison of the receptor affinities of the ET's and SRTX's in rats and atria (ET.sub.A) or cerebellum and hippocampus (ET.sub.B), indicate that SRTX-c is a selective ET.sub.B ligand (Williams, D. L., et al, Biochem. Biophys. Res. Commun., 175:556 (1991)) . A recent study showed that selective ET.sub.B agonists caused only vasodilation in the rat aortic ring, possibly through the release of EDRF from the endothelium (ibid) . Thus, reported selective ET.sub.B agonists, for example, the linear analog ET[1,3,11,15-Ala] and truncated analogs ET[6-21, 1,3,11,15-Ala], ET[8-21,11,15-Ala], and N-Acetyl-ET [10-21,11,15-Ala] caused vasorelaxation in isolated, endothelium-intact porcine pulmonary arteries (Saeki, T., et al, Biochem. Biophys. Res. Commun. 179:286 (1991)). However, some ET analogs are potent vasoconstrictors in the rabbit pulmonary artery, a tissue that appears to possess an ET.sub.B y, nonselective type of receptor (ibid).
Plasma endothelin-1 levels were dramatically increased in a patient with malignant hemangioendothelioma (K. Nakagawa et al, Nippon Hifuka Gakkai Zasshi, 1990, 100, 1453-1456).
The ET receptor antagonist BQ-123 has been shown to block ET-1 induced bronchoconstriction and tracheal smooth muscle contraction in allergic sheep providing evidence for expected efficacy in bronchopulmonary diseases such as asthma (Noguchi, et al, Am. Rev. Respir. Dis., 1992, 145 (4 Part 2), A858).
Circulating endothelin levels are elevated in women with preeclampsia and correlate closely with serum uric acid levels and measures of renal dysfunction. These observations indicate a role for ET in renal constriction in preeclampsia (Clark B. A., et al, Am. J. Obstet. Gynecol., 1992, 166, 962-968).
Plasma immunoreactive endothelin-1 concentrations are elevated in patients with sepsis and correlate with the degree of illness and depression of cardiac output (Pittett J., et al, Ann Surg., 1991, 213(3), 262) .
In addition the ET-1 antagonist BQ-123 has been evaluated in a mouse model of endotoxic shock. This ET.sub.A antagonist significantly increased the survival rate in this model (Toshiaki M., et al, 20.12.90. EP 0 436 189 A1).
Endothelin is a potent agonist in the liver eliciting both sustained vasoconstriction of the hepatic vasculature and a significant increase in hepatic glucose output (Gandhi C. B., et al, Journal of Biological Chemistry, 1990, 265(29), 17432). In streptozotocin-diabetic rats there is an increased sensitivity to endothelin-1 (Tammesild P. J., et al, Clin. Exp. Pharmacol. Physiol., 1992, 19(4), 261). In addition increased levels of plasma ET-1 have been observed in microalbuminuric insulin-dependent diabetes mellitus patients indicating a role for ET in endocrine disorders such as diabetes (Collier A., et al, Diabetes Care, 1992, 15(8), 1038).
ET.sub.A antagonist receptor blockade has been found to produce an antihypertensive effect in normal to low renin models of hypertension with a time course similar to the inhibition of ET-1 pressor responses (Basil M. K., et al, J. Hypertension, 1992, 10(Suppl 4), S49). The endothelins have been shown to be arrhythmogenic, and to have positive chronotropic and inotropic effects, thus ET receptor blockade would be expected to be useful in arrhythmia and other cardiovascular disorders (Hah S.-P., et al, Life Sci., 1990, 46, 767).
The widespread localization of the endothelins and their receptors in the central nervous system and cerebrovascular circulation has been described (Nikolov R. K., et al, Drugs of Today, 1992, 28(5), 303-310). Intracerebroventricular administration of ET-1 in rats has been shown to evoke several behavioral effects. These factors strongly suggest a role for the ETs in neurological disorders. The potent vasoconstrictor action of ETs on isolated cerebral arterioles suggests the importance of these peptides in the regulation of cerebrovascular tone. Increased ET levels have been reported in some CNS disorders, i.e., in the CSF of patients with subarachnoid hemorrhage and in the plasma of women with preeclampsia. Stimulation with ET-3 under conditions of hypoglycemia have been shown to accelerate the development of striatal damage as a result of an influx of extracellular calcium. Circulating or locally produced ET has been suggested to contribute to regulation of brain fluid balance through effects on the choroid plexus and CSF production. ET-1 induced lesion development in a new model of local ischemia in the brain has been described.
Circulating and tissue endothelin immunoreactivity is increased more than twofold in patients with advanced atherosclerosis (A. Lerman, et al, New England J. Med., 1991, 325, 997-1001). Increased endothelin immunoreactivity has also been associated with Buerger's disease (K. Kanno, et al, J. Amer. Med. Assoc., 1990, 264, 2868) and Raynaud's phenomenon (M. R. Zamora, et al, Lancet, 1990, 336, 1144-1147). Likewise, increased endothelin concentrations were observed in hypercholesterolemic rats (T. Horio, et al, Atherosclerosis, 1991, 89, 239-245).
An increase of circulating endothelin levels was observed in patients that underwent percutaneous transluminal coronary angioplasty (PTCA) (A. Tahara, et al, Metab. Clin. Exp., 1991, 40, 1235-1237, K. Sanjay, et al, Circulation, 1991, 84(Suppl. 4), 726).
Increased plasma levels of endothelin have been measured in rats (T. J. Stelzner, et al, Am. J. Physiol, 1992, 262, L614-L620) and individuals (T. Miyauchi, et al, Jpn. J. Pharmacol., 1992, 58, 279P, D. J. Stewart, et al, Ann. Internal Medicine, 1991, 114 464-469) with pulmonary hypertension.
Elevated levels of endothelin have also been measured in patients suffering from ischemic heart disease (M. Yasuda, et al, Amer. Heart J., 1990, 119 801-806, S. G. Ray, et al, Br. Heart J., 1992, 67, 383-386) and either stable or unstable angina (J. T. Stewart, et al, Br. Heart J., 1991, 66, 7-9).
Infusion of an endothelin antibody 1 h prior to and 1 h after a 60 minute period of renal ischaemia resulted in changes in renal function versus control. In addition, an increase in glomerular platelet-activating factor was attributed to endothelin (A. Lopez-Farre, et al, J. Physiology, 1991, 444, 513- 522). In patients with chronic renal failure as well as in patients on regular hemodialysis treatment mean plasma endothelin levels were significantly increased (F. Stockenhuber, et al, Clin. Sci. (Lond.), 1992, 82, 255-258). In addition it has been suggested that the proliferative effect of endothelin on mesangial cells may be a contributing factor in chronic renal failure (P. J. Schultz, J. Lab. Clin. Med., 1992, 119, 448-449).
Local intra-arterial administration of endothelin has been shown to induce small intestinal mucosal damage in rats in a dose-dependent manner (S. Mirua, et al, Digestion, 1991, 48, 163-172). Administration of endothelin-1 in the range of 50-500 pmol/kg into the left gastric artery increased the tissue type plasminogen activator release and platelet activating formation, and induced gastric mucosal haemorrhagic change in a dose dependent manner (I. Kurose, et al, Gut, 1992, 33, 868-871). Furthermore, it has been shown that an anti-ET-1 antibody reduced ethanol-induced vasoconstriction in a concentration-dependent manner (E. Masuda, et al, Am. J. Physiol., 1992, 262, G785-G790). Elevated endothelin levels have been observed in patients suffering from Crohn's disease and ulcerative colitis (S. H. Murch, et al, Lancet, 1992, 339, 381-384).
TABLE I ______________________________________ Plasma Concentrations of ET-1 in Humans ET Plasma Normal Levels Reported Condition Control (pg/ML) ______________________________________ Atherosclerosis 1.4 3.2 pmol/L Surgical operation 1.5 7.3 Buerger's disease 1.6 4.8 Takayasu's arteritis 1.6 5.3 Cardiogenic shock 0.3 3.7 Congestive heart 9.7 20.4 failure (CHF) Mild CHF 7.1 11.1 Severe CHF 7.1 13.8 Dilated cardiomyopathy 1.6 7.1 Preeclampsia 10.4 pmol/L 22.6 pmol/L Pulmonary hypertension 1.45 3.5 Acute myocardial 1.5 3.3 infarction (several reports) 6.0 11.0 0.76 4.95 0.50 3.8 Subarachnoid hemorrhage 0.4 2.2 Crohn's Disease 0-24 fmol/mg 4-64 fmol/mg Ulcerative colitis 0-24 fmol/mg 20-50 fmol/mg Cold pressor test 1.2 8.4 Raynaud's phenomenon 1.7 5.3 Raynaud's/hand cooling 2.8 5.0 Hemodialysis &lt;7 10.9 (several reports) 1.88 4.59 Chronic renal failure 1.88 10.1 Acute renal failure 1.5 10.4 Uremia before 0.96 1.49 hemodialysis Uremia after hemodialysis 0.96 2.19 Essential hypertension 18.5 33.9 Sepsis syndrome 6.1 19.9 Postoperative cardiac 6.1 11.9 Inflammatory arthritides 1.5 4.2 Malignant 4.3 16.2 hemangioendothelioma (after removal) ______________________________________
Rovero, P., et al, British Journal of Pharmacology 101, pages 232-236 (1990) disclosed various analogs of the C-terminal hexapeptide of ET-1, none of which were reported to be antagonists of ET-1.
Doherty, A. M., et al, Abstract, Second International Conference on Endothelin, Tsukuba, Japan, Dec. 9, 1990, and the published manuscript (J. Cardiovasc. Pharm. 17 (Suppl. 7), 1991, pp. 559-561) disclosed various analogs of the C-terminal hexapeptide of ET-1, none of which exhibited any functional activity.
However, we have surprisingly and unexpectedly found that a series of C-terminal hexapeptide and related analogs of ET-1 are receptor antagonists of endothelin. Additional data for the activity of this series of peptides is found in the following references (W. L. Cody, et al, J. Med. Chem., 1992, 35, 3301-3303., D. M. LaDouceur, et al, FASEB, 1992).